All patients were scanned for IVIM-DKI sequences acquired with 14 b-values (0 to 2500 s/mm2) on a 1.5T MRI. An IVIM-DKI model with a 3D total variation (TV) penalty function was implemented to estimate the precise IVIM-DKI parametric maps. Texture analysis (TA) of the apparent diffusion coefficient (ADC) and IVIM-DKI parametric map was performed and reduced using the chi-square test. These features were fed to an artificial neural network (ANN) for characterization of pancreatic mass subtypes and validated by 5-fold cross-validation. Receiver operator characteristics (ROC) analyses were used to compute the area under curve (AUC). Perfusion fraction (f) was significantly higher (p < 0.05) in pNET than PDAC. The f showed better diagnostic performance for PDAC vs. MFCP with AUC:0.77. Both pseudo-diffusion coefficient (D*) and f for PDAC vs. pNET showed an AUC of 0.73. ADC and diffusion coefficient (D) showed good diagnostic performance for pNET vs. MFCP with AUC: 0.79 and 0.76, respectively. In the TA of PDAC vs. non-PDAC, f and combined IVIM-DKI parameters showed high accuracy ≥ 84.3% and AUC ≥ 0.84. Mean f and combined IVIM-DKI parameters estimated that the IVIM-DKI model with TV texture features has the potential to be helpful in characterizing pancreatic masses.Non-invasive characterization of pancreatic masses aids in the management of pancreatic lesions. Intravoxel incoherent motion-diffusion kurtosis imaging (IVIM-DKI) and machine learning-based texture analysis was used to differentiate pancreatic masses such as pancreatic ductal adenocarcinoma (PDAC), pancreatic neuroendocrine tumor (pNET), solid pseudopapillary epithelial neoplasm (SPEN), and mass-forming chronic pancreatitis (MFCP). A total of forty-eight biopsy-proven patients with pancreatic masses were recruited and classified into pNET ( Pancreatic cancer is a challenging malignancy with a 5-year survival rate of only 10% due to its non-specific symptoms at an early stage ,3,4. On Several diagnostic tools, such as the serum levels of carbohydrate antigen (CA 19-9), have been used to characterize pancreatic masses . Howeverb-values)  mm2/s, f = [0 1], and k = [0 3] and was initialized with D = 1.3 × 10−3 mm2/s, D* = 13 × 10−3 mm2/s, f = 0.3, and k = 0.7 based on previous studies [The IVIM-DKI signal was modelled using two models with 14 MA, USA) . A trust studies ,30.b-values for one representative patient are presented in b-value = 0 s/mm2 signal and Equation (2) shows an association between signal variation and b-values in the IVIM-DKI sequence [The IVIM-DKI images at different sequence :(2)S/S0=1 norm of discrete gradient of the map [b-values combinations and consistently improved parameter estimations at different field strengths [The IDTV model can remove any abrupt change in parameter estimates during the optimization process. The TV penalty function was used to iteratively balance out the estimated parameter . The TV  the map . The thrtrengths ,31. The trengths .2 and ROI was verified by the presence of hyperintensity and hyperintensity on corresponding slices of IVIM-DKI at b = 2500 s/mm2 and ADC map, respectively. Mass ROIs were defined on all matching slices of IVIM-DKI images, using ADC maps and conventional sequences such as T2-weighted, contrast enhanced T1-weighted, and DWI images as a reference.A radiologist with more than three years of expertise in abdominal imaging performed the region of interest (ROI) analysis, which was confirmed by another radiologist with more than ten years of experience in abdominal MR imaging and blinded to the reference standard. A free hand ROI was drawn over the lesion to cover the whole mass using MRICron software , as showThe chi-square test of independence was used to select the top ten texture features with the highest feature importance. These top ten features were fed into a 5-layered artificial neural network (ANN) classifier model to differentiate between PDAC and non-PDAC masses. We wanted to investigate the role of an ANN, the most basic form of deep neural network, in classification using IVIM-DKI parametric maps because it has shown potential in screening pancreatic cancer . Bayesiat-test and chi-square test. The ADC and IVIM-DKI parameters  were tested for normality using the Kolmogorov–Smirnov test. The goodness of fit in the standard and IDTV model was assessed using mean adjusted R2 in tumor ROI, with a high value of adjusted R2 indicating that the model adequately reflects the data. The coefficient of variation (CV: standard deviation of ROI × 100/mean of ROI) of the IVIM-DKI parameters estimated using standard and IDTV models in pancreatic masses was measured to evaluate the precision of model estimations, where low CV indicates precise estimation of parameters by a model. The Wilcoxon signed-rank test was used to determine significant differences between mean adjusted R2 of the two comparative models. A Kruskal–Wallis test with a Tukey–Kramer multi-comparison test was used to compute comparison between IVIM-DKI parameters for subtypes of pancreatic masses . The diagnostic performance of a IDTV model was assessed using receiver operating characteristics (ROC) analysis with cut-off values, area under the ROC curve (AUC), sensitivity, and specificity.The patients’ mean age and gender distribution were compared using a two-sample p-value was small, the feature was considered as an important feature and contributed to one of the significant characteristics in classification; otherwise, it was disregarded if the feature had a large p-value and was not considered as an important feature. Thus, the top ten texture features were chosen to improve performance and simplify the complexity of the classifier model. Performance metrics were estimated such as accuracy, precision, recall, specificity, accuracy, F1 score, and classification error on test data from each stratified fold of the 5-fold cross-validation  and this cross-validation was repeated 100 times. A test set from each cross-validation was used to evaluate the performance of the ANN in the classification of PDAC vs. non-PDAC. This 5-fold cross-validation was repeated 100 times; thus, the result was 100 different performance metrics. This validation technique ensures that the data of each patient appeared in the test set a total of 100 times. All statistical analyses were performed using MATLAB .Important texture features were computed using the chi-square test of independence. The chi-square test of independence was used to determine the relationship between feature variables (texture features derived from ADC or IVIM-DKI parameters) and target variables (class labels: pancreatic masses). If the n = 4; age = 29.5 ± 5.1 years old; female:male = 3:1), MFCP , pNETs , and PDAC . p < 0.05) than patients with SPENs and pNETs. Gender differences in pancreatic mass subtypes were significant (p < 0.05) between patients with SPEN vs. PDAC and PDAC vs. pNET. No gender differences between patients with and SPEN vs. pNET were observed. Tumor volume size was estimated for each pancreatic mass: SPEN = 52.14 ± 51.77 cm3, MFCP = 22.98 ± 17.05 cm3, pNET = 8.73 ± 8.02 cm3, and PDAC = 33.43 ± 29 cm3.Based on the reference standard, the pancreatic masses were composed of SPENs  for the IDTV model with R2 = 0.95 ± 0.04 compared to the standard model with R2 = 0.78 ± 0.16. The goodness of fit was significantly higher (p > 0.05); however, f values did demonstrate significant differences between pancreatic masses as shown in p < 0.05) was considerably higher in the pNET as compared to the PDAC  were calculated for each mass. The features of all IVIM-DKI parameters were combined to form a 120  texture-feature set. Similarly, ADC and IVIM-DKI parametric maps’ texture features were combined to create a total of 150 feature sets. These features were then reduced by selecting only high importance scores which were calculated using the Chi-square test and only the top ten features were selected to reduce the model complexity for PDAC vs. non-PDAC pancreatic masses.In PDAC vs. non-PDAC, an ANN with all the features of the combined IVIM-DKI parameters showed the highest accuracy of 90.5%, and AUC of 0.92 as shown in MRI can aid in the differential diagnosis of pancreatic masses which is otherwise a very challenging task. With the assumption of a non-Gaussian distribution of water movement, IVIM-DKI can non-invasively measure diffusion and perfusion parameters to capture the tumor heterogeneity. The efficiency of the novel IVIM-DKI model with a total variation penalty function for characterizing pancreatic mass subtypes was evaluated. f was observed to be useful in distinguishing pancreatic masses. In the differential diagnosis of pancreatic masses, f was found to be a crucial indicator in effectively differentiating PDAC from MFCP and both D* and f for differentiating PDAC from pNET. Further, ADC and D could distinguish pNET from MFCP with good accuracy and AUC. In addition, IVIM-DKI parameters were subjected to textural analysis with textural feature reduction and given to the 5-layer artificial neural network for classification of pancreatic masses. PDAC was distinguished from non-PDAC by texture features of f and combined IVIM-DKI parameters with good accuracy and AUC. IVIM-DKI with the IDTV model was able to remove noise from the parametric maps and produce precise parameter values with low CV. These results demonstrate that the IVIM-DKI model with a spatially constrained optimization method may provide true underlying texture features for successful characterization of pancreatic masses.A new approach was used to precisely measure IVIM-DKI parameter values to differentiate between pancreatic masses. This IDTV model has been effectively applied in characterization of prostate lesions  and patiAlthough the efficacy of IVIM-DKI in pancreatic mass characterization has yet to be completely examined, it provides a quantitative evaluation of perfusion and heterogeneity in the tumor. Mean f was able to characterize PDAC from pNET, where f was high in the latter, which might be attributed to the large amount of desmoplastic stroma and poor tumor vascularity in PDAC ,37,38,39Additionally, this study estimated cut-off values for further subtyping of pancreatic masses. ADC and D showed good accuracy and AUC for characterizing pNET vs. MFCP. The presence of granulation and fibrosis in MFCP  may haveChronic pancreatitis of the mass-forming type has an imaging appearance like PDAC, leading to misdiagnosis on cross-sectional imaging. On CT perfusion imaging (CTP), blood volume (BV) and blood flow (BF) have been shown to differentiate PDAC from MFCP ,45. PerfIn assessing pancreatic masses, whole-volume TA was also carried out for PDAC vs. other masses. TA can offer information on non-invasively detecting small variations in the intensity of a lesion location and can yield additional information on microscopic tissue heterogeneity . This stb-values for a shorter acquisition duration. Fourth, the correlation between IVIM-DKI parameters and histopathological markers were not assessed in the current study. Fifth, the IDTV model was not compared with Bayesian techniques since it was outside the scope of this study. Lastly, the texture analysis comparing all possible subtypes of pancreatic masses was not performed because the sample size was insufficient to provide conclusive evidence for subtypes analysis; however, data augmentation techniques were used for this limitation, but results were found to be over-biased and inconclusive due to very small dataset for MFCP and SPEN.This study has a few limitations including, first, the study cohort was small, and the results from analysis might be biased due to it being a single center study and thus conclusion can be considered as preliminary evidence requiring wider validation. Further, this study needs to be performed with large multicentric data analysis. Second, in this cohort, age and gender were not matched; however, no correlation between age or gender distribution with ADC or IVIM-DKI parametric values were observed. Third, the acquisition time to acquire IVIM-DKI was 16 min, which might cause motion artifacts; hence, future investigations will be required to optimize the A larger dataset of rare pancreatic cancers such as SPEN and MFCP needs to be acquired so that this can help us in understanding its contribution in characterization of pancreatic masses. Machine learning-based texture analysis may benefit not only in the accurate diagnosis and management of cancer, but also in the virtual reality-based surgical procedure planning ,50. ThisIn conclusion, this study demonstrated that the IVIM-DKI model with the spatial regularization optimization approach has a strong potential to be used as an additional paradigm for differential diagnosis of pancreatic masses. Perfusion measures such as D* and f were found to out-perform with accuracies of 88% and 77%, respectively in discriminating pancreatic adenocarcinoma from pancreatic neuroendocrine tumor and mass forming-chronic pancreatitis than the diffusion measures (ADC and D). Non-invasive characterization of pancreatic masses can also largely benefit from whole-volumetric texture analysis of f and combined texture features from IVIM-DKI parameters paired with machine learning-based classification with accuracies of 84.9% and 84.3%, respectively.


The increasing growth of electronic information science and technology has triggered the renaissance of the artificial sensory nervous system (SNS), which can emulate the response of organisms towards external stimuli with high efficiency. In traditional SNS, the sensor units and the memory units are separated, and therefore difficult to miniaturize and integrate. Here, we have incorporated the sensor unit and the memory unit into one system, taking advantage of the unique properties of the ion-gel system. Meanwhile, the weaving-type memory array presents paramount advantages of integration and miniaturization and conformal lamination to curved surfaces. It is worth noting that the electrical double layer (EDL) within the ion gel endow the device with a low operation voltage (<1 V) to achieve low energy consumption. Finally, according to the relationship of pressure stimuli and electrical behavior, the integrated responsiveness-storage external stimuli ability is achieved. Our work offers a new platform for designing cutting edge SNS. To develop an artificial sensory nervous system, we use an ion-gel system and solve the issue that sensor units and memory units are separated and difficult to miniaturize and integrate. Consequently integrated responsiveness-storage external stimuli ability is achieved. To mimic the functions of organisms for reacting to external stimuli and storing the sensed signal, integrated memory-sensing systems have sprung up. For example, Bowen Zhu et al.9 have integrated a pressure sensor into a memory device to establish a bridge between the state-of-the-art E-skin devices and skin-inspired-integration haptic memory devices to mimic the haptic memory of natural skin. In another instance, with respect to visual-memory systems, integrating UV light sensors onto the memristor array was capable of affording an essential exteroceptive sensory memory for cognitive tasks.10,11Resistive switching-based memristors (RS), acting as a fascinating emerging storage technology, have received much attention due to theirAlthough these works may represent significant steps towards the realization of bio-inspired SNS, in these previous memory-sensing systems, the memory units and sensing units were independent and were not beneficial for the integration and miniaturization. Incorporating the memory units and sensing units into one system involves material selection, matched operation conditions and so on.12–14 As a consequence, stretchable memristors have emerged to address the issue of implantable and comformal applications. With the springing up of fiber-based electronics and wearable electronic systems, functional fibers provide a promising platform for flexible and wearable memristors for computing and storage applications.15,16To solve the above issues, we present a memory-array based iontronic system, taking advantage of the stimuli-responsive characteristic of ions within this iontronic system. Specifically speaking, the iontronic can play a dual role: inductive stimulus and storing charges (as an insulator layer of a memristor). Meanwhile, for biomedical applications, such as implanted treatment and human–machine interface, it is highly desired to explore devices with excellent flexibility and mechanical compatibility with biological tissues.2O3 as a resistance switching layer and carbon fiber as the counter-electrode. However, the driving voltages of their devices are not relatively low, and the ON/OFF ratio was not high (∼103). To alleviate the high energy consumption from a high operating voltage, the iontronics have been newly emerging, with the interdisciplinary concept that bridges electronics and ionics. Within the iontronics systems, the EDLT can realize the low-voltage operation to promote energy efficiency.17–19 The ionic systems utilized in electronics have been reported. Guoyun Gao et al. have developed a triboiontronic transistor of MoS2 with electric double layers (EDLs) formed in electrolyte-gated field-effect transistors (FETs), inducing an extremely large local electric field that gave a highly efficient charge carrier control in the semiconductor channel.20 The resistance random-access memory (ReRAM) could also be adopted iontronics, in which electrical switching effects between the on and off states are triggered by ionic motion in the oxide devices.21Anjae Jo has reported a textile resistance switching memory based on Al-coated threads with a native layer of AlIn this manuscript, we, for the first time, have integrated the iontronic system into a wearable memristor electronic system to render the integration of stimuli-responsiveness-memory. The low-voltage operation has been achieved by the EDLT in ion-gel to considerably decrease energy consumption. In particular, we introduce the brain-inspired device technologies into the non-volatile resistive memory array. The device shows bidirectional continuous weight modulation behaviour. These experimental results consolidate the feasibility of sensing-memory integrated systems and pave a new avenue for constructing energy efficient and large-scale neuromorphic systems.22.122,23e.g., Pt, Au electrode) by magnetron sputtering. Then, the ion gel (composed of 1-ethyl-3-methylimidazolium bis(trifluoromethyl sulfonyl)imide ([EMIM][TFSI]) ion liquid, poly(ethylene glycol)diacrylate (PEGDA) monomers, and 2-hydroxy-2-methylpropiophenone (HOMPP) photo-initiator in a weight ratio of 90 : 7 : 3)19 were patterned on the PEDOT:PSS aligned with the Pt/Au electrode by photolithography, followed by kinking the active electrode (Ag electrode)-coated cotton fibers in a horizontal orientation. It is well known that the quality of the electrode is the prerequisite for an excellent performance of devices. Therefore, the metal-coated fibers are characterized firstly. Fig. S124 ion-intercalation mechanism,25,26 phase mechanism,27 and ion control mechanism28,29The Ag-coated cotton fibers and Pt-coated cotton fibers are knitted with a vertical-horizontal cross configuration and the semiconductor and dielectric layer is sandwiched between them (as shown in 2.27 Ω) requires an electroforming process with the application of a forming voltage around 0.05 V to activate the bidirectional RS behaviour. The maximum value of nonlinearity is 107 under the voltage of 0.5 V and a steep variation in the resistance from the initial high-resistance state to a low-resistance state, only needing a small positive voltage of about 0.02 V. In particular, the threshold voltage to open the device is much lower (could operate at a low gate voltage) due to the ultrahigh capacitance of the ion gel gate (6–7 μF cm−2), which considerably decreases the power consumption. The power consumption is about 5 × 10−8 J. The relatively lower operation voltage stems from the extremely high capacitance of the ion gel as the dielectric material17–19,30 and considerably decreases energy consumption. This also demonstrates the iontronic system in the development of electronics technology with low power consumption. The device shows bipolar resistor changing behaviour due to the bipolar transport characteristic of the semiconductor PEDOT. To validate the bidirectional TS behaviour, the sweep path of 0 → −0.5–5 → 0 → 0.5–5 → 0 V was operated on the device. Fig. SI2Prior to investigating the properties of the memristor matrix array, the electrical performance of the single unit was firstly explored. I–V curve of device under varied ion gel recipes composed of 1-ethyl-3-methylimidazolium bis(trifluoromethyl sulfonyl)imide ([EMIM][TFSI]) ion liquid, poly(ethylene glycol)diacrylate (PEGDA) monomers, and 2-hydroxy-2-methylpropiophenone (HOMPP) photo-initiator in a weight ratio of 1.72 : 0.4 : 0.12 (pink curve) and 4.5 : 0.4 : 0.12 (blue curve). This is clearly shown in Fig. SI4,To illustrate the role of the ion gel in the device, the device with Pt electrode/PEDOT/Ag electrode structure but in the absence of ion gel is measured as a comparative experiment. As shown in Fig. SI3,I–t curve is displayed in Fig. SI531,32The device behaviour under a pulse voltage also can be served as a characteristic index for reflecting the relaxation properties. The pulse voltage posed on the device and the corresponding I–V curve of the device under varied complement currents of 0.1 mA, 0.05 mA, 0.01 mA, and 0.005 mA. The ON/OFF is almost the same as varied complement currents but with sacrificing the hysteresis. Based on the evolution of current–voltage (I–V) characteristics as a function of complement currents, the maximum allowed complement currents for reliable operation for devices are about 10−3 A. Fig. SI 7I–V curves of the device under a varied sweep range , the different shapes of these curves demonstrate that this device can tolerate voltages with a large range.It is widely known that the complement current is also an important parameter to determine the performance of the device. Fig. SI6I–V performance of the memory device in the initial cycle, 10 cycles, 20 cycles, and 200 cycles. The rough overlap of every curve demonstrates its pronounced endurability. The device can withstand an endurance of over 200 cycles under the 100 μs/2 V programming pulses and 100 μs/0.3 V read pulses, with a 1.8 ms wait time between each program and read pulse  and for four months (blue line). It can be clearly observed that the properties of the as-prepared device are almost identical to those of the pristine state and stayed in an ambient environment for four months later.The device also possesses excellent environmental stability. Fig. SI92.3I–V curves of the device under varied bending degrees . It can be seen in −1 to 38.7 m−1, the current increases from 13.8μA to 6820μA at a given voltage of 0.1 V, and the corresponding resistance decreases from 7246 Ω to 1.47 × 10−5 Ω. The higher bending degree leads to higher conductivity (relatively low resistance). This can be explained by the fact that as the device is in the bending state, the capacitance of the ion-gel increases and leads to an increase in conductivity. I–V curve of the device  under a bending degree of 38.71 cm−1. The device shows excellent bending flexibility without sacrificing cycle stability. The stability of the device in a repetitive bending state indicated its promising flexible wearable applications. Apart from bending deformation, compressing deformation is also studied. The typical I–V curve of the device under varied pressure  is shown in −4 Ω to 2.76 × 10−4 Ω. Similar to the pretext bending deformation, this can also be ascribed to the fact that as the device is in a loaded state, the capacitance of ion-gel increase and lead to an increase of conductivity. I–V curve of the device under varied pressure. The almost overlap of curves at different pressure indicates its superior endurability in the compressing state (under the loaded state of 50 kPa). The device shows excellent bending flexibility without sacrificing cycle stability. Fig. S10I–V curve of the device with free-standing state, compressing state (50 kPa), and recovery state after experiencing pressure. As shown in Fig. S10,As aforementioned in the pretext, the iontronic system endows the device with stimuli-responsiveness. The characteristics of the integrated memory-sensing device under deformation have been systematically investigated. 2.433 The wearable electronic textiles are subjected to extensive mechanical stress and deformation, which might induce the separation or the shift of contact position between threads. With respect to the mechanism of the typical Ag-based (Ag electrode as the active electrode) two-terminal memristor, Ag filament-mechanism is relatively common.34–37 However, due to the thickness of the ion gel reaching 100 μm, the Ag conductive filaments are not able to diffract to the counter electrode. We anticipated that the mechanism of this device was not Ag conductive filament mechanism. To verify our hypothesis, TEM is applied.However, the EDLT produced by ion gel has dual functions of electrochemical doping and electrochemical dynamic. What mechanism plays the predominant role? Or whether both the Ag filament mechanism and electrochemical dynamic coexist? So, we constructed a device with the Au-EDLT-Pt (in the absence of Ag filament-mechanism) for a comparison. If the properties of the device distinguish from the counterpart of Ag-EDLT-Pt, the Ag filament mechanism may play a vital role. Otherwise, if the properties are the same as the counterpart of Au-EDLT-Pt, the mechanism is not based on Ag filament-mechanism. To get insight into the mechanism, the device to be studied adopted a coplanar structure. 2, 400 μm2, 1000 μm2) are nearly the same, demonstrating that the mechanism of this device was not Ag conductive filament mechanism, which was strongly associated with the area of the electrode.38 To further demonstrate the non-Ag-conductive filament mechanism, the Au electrode replaced the Ag electrode to obtain the device Pt/PEDOT:PSS-ion gel/Au as a comparative experiment. The typical I–V curve of the device Pt/PEDOT:PSS-ion gel/Au is shown in Fig. SI11.The transmission electron microscope (TEM) is a powerful tool for investigating the mechanism of memristor devices. A positive bias pulse poses on the as-prepared device to trigger the device to its on-state. The TEM is carried out as the device is kept in its on-state. As shown in I–V curve of the device with P3HT as active material of memristor under varied sweep range is shown in Fig. S126) and low-voltage operation.To demonstrate the universality of this fiber-type memristor, the P3HT was used to replace PEDOT as an active material. The typical 2.5Finally, the aforementioned integrated sensing-memory fiber-type memristor was extended to an 8*8 array as sensory nervous systems. As shown in 36), good durability (200cycles), considerably surpassing the transitional PEDOT:PSS memristor device . The relationship of resistance vs. pressure turns out to be relatively linear with excellent stability of output under deformation. Our work will provide new insight for the development of the sensing-memory neuromorphic network and significantly promote the chip to achieve high-level integration and miniaturization.In summary, we constructed an integrated fiber-based sensing-memory to achieve high-level integration and miniaturization. The integrated fiber-based sensing-memory is realized by the particular properties of the ion-gel system. Furthermore, the ion-gel system endows the device with low-operation characteristics and a considerable decrease in energy consumption. Meanwhile, the weaving-type memory array presents conformally attached skin. The device turns out to be of bipolar characteristic with an excellent ON/OFF (1044.1The cotton fibres were bought from Hangzhou Yangyang cool company. PEDOT:PSS ink (≥99%), ion liquid 1-ethyl-3-methylimidazolium bis(trifluoromethyl sulfonyl)imide ([EMIM][TFSI]) (≥98%), poly(ethylene glycol)diacrylate (PEGDA) monomers (≥98%), and 2-hydroxy-2-methylpropiophenone (HOMPP) (≥99%) were bought from Admas chemical limited liability company.4.2(1) Fiber-coated electrode fabrication.e.g. Pt, Au electrode, 5 nm/70 nm, Cr/Au and 5 nm/90 nm Cr/Pt) with tightly adhered shadow mask through thermal evaporation methods or magnetron sputtering, followed by dip-coating with PEDOT:PSS ink (1 wt%). Prior to ion gel pattern, PEDOT:PSS ink coated fiber was annealed at 90–110 °C for 1 h.The commercially-available cotton fibers in horizontal orientation selectively coated inertia electrode imide ([EMIM][TFSI]) ion liquid, poly(ethylene glycol) diacrylate (PEGDA) monomers, and 2-hydroxy-2-methylpropiophenone (HOMPP) photo-initiator in a weight ratio of 90 : 7 : 3) were patterned by photolithography , followed by knitting the active electrode -coated cotton fiber in horizontal orientation.(2) The coplanar structure for mechanism investigation through transmission electron microscope TEM.e.g. Pt, Au electrode, 5 nm/70 nm Cr/Au and 5 nm/90 nm Cr/Pt) were evaporated through electron beam evaporating 100 nm Ag and patterned via typical photolithography procedure , followed by metal etching. Ag active electrode was patterned via typical photolithography procedure  and followed by electron beam evaporating 100 nm Ag and lifting off procedure by acetone. PEDOT:PSS patterned by means of ink-jet printing of PEDOT:PSS ink (1%) between the inertia electrodes and active electrodes. The ion gel consisting of 1-ethyl-3-methylimidazolium bis(trifluoromethyl sulfonyl)imide ([EMIM][TFSI]) ion liquid, poly(ethylene glycol)diacrylate (PEGDA) monomer, and 2-hydroxy-2methylpropiophenone (HOMPP) photo-initiator (90 wt%: 7 wt%: 3 wt%) was cast above the patterned PEDOT:PSS. Under UV exposure, polymerization of PEGDA was initiated by the reaction between monomers and radicals originating from HOMPP to produce a cross-linked structure. The unexposed region was washed away using DI water.Si substrate (120 μm) was cleaned under sonication with acetone, isopropanol, and deionized water (DI) water, respectively. The inertia electrodes . X-ray diffraction (XRD) patterns were obtained on a Panalytical X-ray diffractometer with Cu radiation of 2.2. The samples were scanned in the 2θ range of 10–80° with the step size of 0.05. Raman (HORIBA/LabRAM HR Evolution) measurements were performed on a RenishawInVia. Raman microscope was equipped with a 532 nm laser. The electrical performances of the memristor were performed on a Keysight B1500A semiconductor device analyzer. All measurements were carried out under ambient conditions.Yanfang Meng conceived the experiment. Yanfang Meng and Jiaxue Zhu conducted the experiments. Yanfang Meng analysed the results. All authors reviewed the manuscript. All figures were drawn by ourselves.There are no conflicts to declare.NA-004-D1NA00703C-s001


Real-world evidence plays an important role in the assessment of efficacy and safety of novel therapies. The increasing use of immune checkpoint inhibitors (ICIs) in patients with advanced melanoma has led to notably improved clinical outcomes, while they are also associated with immune-related adverse events (irAEs). The majority of the available data are based on clinical trials, where the investigated subjects often do not adequately represent the general patient population of the everyday practice. Although there is a niche of objective biomarkers for the future treatment response of ICIs, certain studies suggest that irAEs may be predictive. The aim of this study was to carry out a retrospective analysis of treatment data from patients with advanced melanoma, treated with a single anti-PD-1 agent (pembrolizumab or nivolumab) during a 77-month-long period. Treatment efficacy and occurrence of adverse events were analyzed to identify potential predictive markers. Primary and secondary endpoints were the overall survival (OS) and progression-free survival (PFS). In our cohort, we demonstrated that the occurrence of more than one irAE showed a correlation with response to PD-1 ICI therapy and improved the OS and PFS. Our study suggests, that the grade of toxicity of the irAE may affect the survival rate. Over the last decade, the treatment of advanced melanoma (AM) has undergone a substantial change. The introduction of immune checkpoint inhibition (ICI) and targeted therapies has substantially improved the clinical outcomes of AM patients ,2,3,4. IFollowing the approval of ipilimumab, novel ICIs targeting programmed cell death (PD)-1 were also approved for the treatment of AM. The programmed death ligand 1 (PD-L1) protein is expressed in the tumor cell membrane ,10,11. UIn addition, PD-1 ICIs were recently also approved for stage III melanoma as a novel adjuvant treatment option. Their administration significantly decreases the hazard ratios of melanoma recurrence compared to placebo treatment and also ipilimumab . Ipilimumab and nivolumab can be also given in combination therapy, which provides a higher objective response rate. However, the safety profile of this combination is less favorable and it does not result in a significant survival benefit compared to PD-1 ICI monotherapy ,8. BRAF and MEK inhibitors are small molecules that are used in combination treatment, which can offer a survival benefit for around the half of those AM patients who have an activating BRAF mutation . While tNovel immunotherapies for AM are approved based on the clinical outcomes of phase III studies, which require carefully determined inclusion and exclusion criteria, e.g., patients with an ECOG (Eastern Cooperative Oncology Group) status of ≥2, and those with active brain metastases or autoimmune diseases were excluded. However, the majority of patients in the everyday practice with AM do not fit these strict eligibility criteria. Moreover, significant differences in the median OS were reported in patients who met these eligibility criteria, in contrast to those patients who did not (18.3 vs. 5.43 months) . Thus, tHere, we evaluated the efficacy and safety of an out-of-trial, real-life population of AM patients treated with anti-PD-1 monotherapy with nivolumab or pembrolizumab. A retrospective descriptive analysis of patients with unresectable stage III (M0) or stage IV melanoma (M1a–d) who were undergoing treatment with a single PD-1 inhibitor agent, pembrolizumab or nivolumab, at our department was carried out. We analyzed a 77-month period from 1 May 2015 to 31 October 2021. We collected patient data from our electronic health records .Inclusion criteria were the following: the presence of metastatic or unresectable cutaneous AM that was treated with PD-1 ICI monotherapy during the study period, for at least two cycles of the standard dosing of nivolumab (3 mg/kg every 2 weeks) or pembrolizumab (2 mg/kg every 3 weeks). Exclusion criteria were patients with primary uveal melanoma and primary mucosal melanoma, and patients with incomplete medical records. The data cutoff was set to 30 June 2021, giving a minimum follow-up of four months. The patients received either nivolumab or pembrolizumab administrated intravenously at our outpatient clinic. The dosage of nivolumab was 3 mg/kg every second week, or a 240 mg flat dose every two weeks, or a 240 mg flat dose every four weeks. The dosage of pembrolizumab was 2 mg/kg every three weeks, or a 200 mg flat dose every three weeks, or a 400 mg flat dose every six weeks. The choice of PD-1 ICI and its dosage was selected based on the availability of the drug and the Hungarian AM treatment guideline, that changed during the investigated period given its long duration. PD-1 ICI treatment was administered until disease progression, unacceptable toxicity, death of the patient, or if the treating physician decided to withdraw the drug. Patients who had received prior treatment with BRAF/MEK inhibitors, prior treatment with whole brain radiotherapy (WBRT), or superficial radiation therapy were also included. The patients’ demographic data, subtype of the primary melanoma, disease stage at first presentation, BRAF gene mutation status and lactate dehydrogenase (LDH) level data were collected from the electronic health records, as well as baseline characteristics before PD-1 ICI therapy initiation. The treatments used, their duration, efficacy based on imaging  and clinical evaluation, and the reasons for treatment cessation were analyzed. Data collection for the analysis of treatment-related AEs including the grade of severity and type of AE was carried out based on the common terminology criteria for adverse events (CTCAE) classification v.5.0. p value <0.05 was considered statistically significant. All analyses have been conducted with the IBM SPSS Statistics for Windows software, Version 25.0. .We determined real-world OS (rwOS) based on the start date of anti-PD-1 therapy and the date of the demise or last follow up of the patient. Real-world progression-free survival (rwPFS) was calculated from the start of the treatment until disease progression or death, or, if there was no progression, until the last follow-up date. The Mann–Whitney U test or Fisher’s exact test was used for descriptive analysis, as applicable. RwOS and rwPFS rates were estimated using the Kaplan–Meier method and differences between survival curves for each risk factor were compared by log-rank test. Parameters significant in univariate evaluation were selected for multivariate Cox proportional hazard regression analysis. The CI was set to 95% and a At the start of data collection, 126 patients treated at our department with a single agent PD-1 ICI were identified. Three patients had ocular melanoma and four patients had mucosal melanoma and were excluded from the analysis. Consequently, 119 patients were analyzed, who were treated with the PD-1 ICI nivolumab or pembrolizumab between 1 May 2015 and 31 October 2021. Demographic and clinical characteristics at baseline are detailed in The histopathological characteristics of the primary melanoma were as follows: 36 patients (30.3%) had pT4b melanoma and 35 patients (29.4%) had pTx (in which the Breslow score of the primary tumor cannot be assessed) or occult melanoma. Based on tumor-infiltrating lymphocytes (TIL) within the primary melanoma, we found 15 (12.6%) were brisk and 74 (62.2%) remained unknown. The nodular melanoma subtype was identified in 33 cases (27.7%) and the superficial spreading melanoma subtype and those not classified made up 23 cases (19.3%). A total of 98 patients (82.32%) had primary cutaneous melanoma and 21 patients (17.65) had occult primary melanoma. Twenty-five patients (21%) had brain metastases at the time of treatment initiation .Eighty-one patients  had BRAF wild-type melanoma, whereas only 31 (26.1%) patients harbored the BRAF V600 mutation. Only seven (5.88%) patients with BRAF-positive melanoma received nivolumab or pembrolizumab PD-1 ICI as first-line treatment . A totalThe objective response rate (ORR), defined as the rate of patients with a complete response (CR) and partial response (PR), was only 16.8%. The disease control rate (DCR), defined as rate of patients with CR, PR and stable disease (SD), was 52.08% . The treThe ORR for BRAF wild-type patients was 19.31%, and the ORR for BRAF mutant patients was only 9.67. However, from 31 patients with a BRAF V600 mutation, only seven received first-line anti-PD-1 therapy. Among the patients harboring a BRAF mutation, CR occurred in two cases and PR only in one case.n = 20), those AM patients who achieved an objective response; and non-responders (n = 89), patients who achieved SD or PD as the best response, were compared. Outcome data for one patient was not available (NA)  . The assble (NA) .The whole population median OS was 130 weeks (30 months) a and thep = 0.982)  .p = 0.004, p = 0.011)  c,d. p = 0.022)  and received the PD-1 ICI not as first line therapy, had a longer survival compared to patients with a BRAF mutation and no previous BRAF inhibitor treatment  . p = 0.880, median PFS 61 weeks vs. 36 weeks; HR for progression 1.62, 95 CI 0.18–14.25, p = 0.666 That patients who had no brain metastases had a longer OS and PFS compared to those who had brain metastases  had a longer OS and PFS compared to those who did not have an irAE .The presence of a grade 3–4 irAE meant a better outcome   occurred in a total of 55 patients (46.2%). A mild irAE (grade 1–2) developed in 37 patients (31.08%), while a severe irAE (grade 3–4) developed in 18 patients (15.12%) . There wThe present study has been conducted at a single center between 2015 and 2021, and includes the retrospective data analysis of patients with AM treated with anti-PD-1 monotherapy with nivolumab or pembrolizumab. This cohort consisted of 119 patients with known unfavorable characteristics such as brain metastases (stage M1d disease), being older individuals (≥70 years old), poor performance status (≥2 ECOG), prior treatment, BRAF V600 mutation and the presence of an active autoimmune disorder requiring systemic steroid treatment. The majority of these patients would not meet the inclusion and exclusion criteria of the respective phase III trials .In our real-world study, the median OS was 30 months (130 weeks), compared with 37.3 months in the Checkmate-066 phase III trials on anti-PD-1 antibodies, and 32.7 months in the Keynote-006 study, respectively. However, our patients presented with a 12.5-month (54 weeks) median PFS, which was better than the results of the phase III trials (5.1 and 8.4 months) ,17,18,19On the background of the prolonged PFS and low ORR, the small number of patients, who reached CR (6.7%) or PR (10.08%) developed a long-lasting immune response against the tumor, expecting a long-term survival; this also explained why the median prolonged OS was not reached during this study period.As expected, patients with negative prognostic factors, such as elevated LDH levels, poor performance status (ECOG PS ≥ 2), or brain metastases (stage M1d), may benefit less from PD-1 inhibition. However, in our cohort, the majority of patients had an ECOG PS 0–1 , only two patients had an ECOG performance status ≥ 2, and nine (7.56%) patients had an elevated LDH level at the beginning of the anti-PD-1 therapy. Therefore the correlation of the outcome was not significant and explained the prolonged PFS. Untreated melanoma brain metastases had a poor prognosis  ,22. Our We also investigated whether the administration of anti-PD-1-ICI as a first-, second-, or third line therapy influenced the survival outcome. We observed that those individuals who received PD-1 ICI as a second- or third line treatment benefited more than those who received PD-1 ICI as a first-line therapy. On the background of this difference, it is possible that those patients who had BRAF-positive AM and were previously given BRAF and MEK inhibitors in combination achieved a fast regression of the tumor burden. However, among the BRAF wild-type AM patients who received PD-1 ICI as a second-line treatment, few of them received ipilimumab as first-line therapy and it took significantly more time to achieve a therapeutic response. The majority of BRAF wild-type AM patients who were treated with second-line anti-PD-1 monotherapy had received chemotherapy as the first-line treatment.According to our results, those patients who harbored BRAF wild-type AM showed a survival benefit when compared to patients with BRAF V600-mutant melanomas. A potential explanation is that patients with BRAF-mutant melanomas were administered BRAF and MEK inhibitor combination therapy or previous BRAF monotherapy (77.4% of the patients with BRAF mutations).Those few patients who did not receive previous treatment with a BRAF-targeted therapy (5.8%), however, showed less favorable survival outcomes. This finding may be explained by the fact these patients had a high tumor burden and already suffered from symptoms of the disease. Thus, patients receiving a first-line therapy with a BRAF and MEK inhibitor combination achieved a rapid decrease in tumor burden and improvement in their symptoms.There is an ongoing debate over whether ICI or a BRAF and MEK inhibitor combination should be given as first-line therapy in BRAF V600 mutant AM and if this decision has a significant impact on the long-term survival of the patient. The findings of recent prospective trials  resulted in a paradigm shift . Both trp = 0.010). There is only limited data on the efficacy of RT in patients receiving anti-PD-1 ICI. In a prospective clinical trial, 25 AM patients receiving a combination of RT and anti-PD-1 ICI had better outcomes at the irradiated and also the non-irradiated areas, that could be explained by the abscopal effect [Those patients in our cohort who simultaneously received RT and anti-PD-1 ICI had a higher OS  that are manageable with early administration of systemic corticosteroids or immunomodulators. In the present study, severe irAEs (grade 3 or 4) in 31.08% of patients were reported, while mild irAEs (grade 1 or 2) occurred in only 15.12% of the patients. These results are in line with the observed frequency of irAE seen in clinical trials ,15,28. We found that patients with one or more than one irAE (>1 irAE) had significantly longer OS and PFS compared to patients with no irAE. The grade of toxicity also affected the survival, the patients with a grade 3–4 irAE had a significantly improved PFS. These findings suggest the presence of one or more irAEs is an independent predictive marker of PFS and OS. Suo et al. showed that, at a 12-week timepoint, the development of irAEs during anti-PD-1 ICI was associated with better treatment outcomes. Weber et al.  publisheImportantly, the present study has various major limitations, such as its retrospective nature, that result in significant selection bias. In addition, our study is limited by AE reporting being based on the medical documentation; thus, grade 1–2 irAEs could have been underreported.Despite the different baseline patient characteristics, the anti-PD-1 monotherapy showed encouraging long-lasting responses in patients with AM in a real-world setting, including long-term outcomes with the potential risk for developing an irAE. This retrospective study demonstrated that the occurrence of one or more irAEs was significantly associated with anti-PD-1 therapy response and they have been shown to be an independent predictive marker of PFS and OS.


Preterm-born children are at risk of slower psychomotor development. This risk may be associated with low birth weight and other perinatal factors and morbidities.We aimed to assess psychomotor development in school-aged preterm children, and to determine whether some early motor and perinatal variables could be related to and/or predict the later motor achievements.Parents of 54 very low-birth-weight preterm, 24 extremely low-birth-weight preterm and 96 control children completed the Movement Assessment Battery for Children (MABC-2-C) checklist and were interviewed about the motor milestones of their children.Significant differences were found between the preterm and control groups in the MABC-2-C results. MABC-2-C outcomes were significantly predicted by the age of crawling, the use of steroids, mechanical ventilation and intraventricular hemorrhage (IVH).The use of screening tools may allow the rapid identification of psychomotor development delays. The presence of some perinatal risk factors and some motor milestone attainments could be related to motor development in the later childhood of preterm children. Our hypothesis was that preterm children would score lower than control children on the overall scale and on each subscale. Besides, we aimed to determine whether the current motor development of preterm children could be associated with some early developmental factors, such as motor milestone attainments and perinatal risk factors.2.2.1.The study conducted was observational, descriptive and cross-sectional. Preterm children  were recruited from a cohort of neonates who were born before 37 weeks of gestation, had a birthweight under 1500 g and were admitted to the neonatal intensive care unit (NICU) of the Hospital Universitario Central de Asturias  between January 2009 and December 2011. The inclusion criteria were a gestational age of ≤37 weeks and a birth weight of ≤1500 g. The exclusion criteria were death, no follow-up, preterm children with a diagnosis of malformations and/or congenital anomalies that led to evident neurological alterations, a diagnosis of cerebral palsy or an intelligence quotient (IQ) of ≤70, as determined by the Reynolds Intellectual Screening Test (RIST) test 2.2.The MABC-2-C The Haizea-Llevant Scale The RIST test was used as a screening method for IQ. It consists of two tasks: one for verbal IQ (Guess what) and another one for non-verbal IQ (Odd-item) assessment. Its purpose in the present study was to exclude those children, both preterm and controls, who scored 70 points or less, regarding them as having low cognitive performance that could potentially affect psychomotor development.For the perinatal risk factors analysis, the variables were retrieved from medical records and selected from existing literature on motor development . About t2.3.The primary caregivers whose children fulfilled the inclusion criteria were informed by a letter of the purpose of the research, and they were given the opportunity to participate in the study. Those who accepted to participate in the study provided written informed consent before the study began. Then, a pediatrician interviewed the parents, who completed the questionnaires while the children performed the IQ test applied by a psychologist. The duration of the appointment was approximately one hour.2.4.Analyses were performed using SPSS 19.0 for Windows. Means, standard deviations and percentages were calculated for descriptive data . The Chi-square test, with Cramer's V as a measure of effect size, was used to compare groups on the nominal variables. A Student's t-test was employed to compare the performances of the control group and the preterm children, and ANOVA with a Bonferroni post-hoc test was used to compare the ELBW, VLBW and control groups. Cohen's d for the Student's t-test and Eta-squared for the ANOVA were used to estimate the effect size. Both ANOVA and the Student's t-test were followed by an ANCOVA to control for participants' age and gender. Pearson correlation coefficients were calculated for the motor outcomes in preterm children and milestones and perinatal factors. Significantly correlated variables were included in a stepwise regression model. A p-value lower than 0.05 was considered significant. The data that support the findings of this study are available from the corresponding author upon reasonable request.3.3.1.2,87 = 3.410, p = 0.038, η2 = 0.073) and walking . Post-hoc comparisons revealed that such differences in walking were between the controls and ELBW children (p = 0.011), while the comparisons for crawling did not remain statistically significant.No significant differences were found between the preterm group and the control group in terms of age, sex, maternal education level or laterality, nor did the groups significantly differ in the age (corrected for the preterm group) at which they reached all motor milestones. However, some significant differences were found when comparing the ELBWs and VLBWs with the controls for the ages of crawling , although we did not find significant differences between control and preterm children regarding the number of children in each group who were delayed in the development of each milestone (p > 0.05).We also found significant differences in the number of controls and preterm children who did not crawl  were identified as at risk of having motor difficulties: medium risk in 4.5% (N = 4) and high risk in 14.77% (N = 13). The differences between the percentage of preterm and the percentage of controls that were classified as at-risk were not statistically significant (p = 0.086). Regarding the birth weight, in the VLBWs, 2% (N = 1/54) were classified as at medium risk, and 18.4% as at high risk (N = 9/54); in the ELBWs, 13.6% were labeled as at moderate risk (N = 3/24), and 9.1% as at high risk (N = 2/24). In the control group, 12.5% of the sample reached risk values (N = 12/96), with 6.25% reaching moderate risk (N = 6), and 6.25% reaching high risk (N = 6). No significant differences were found between the risk percentages for the VLBWs and ELBWs (p = 0.715).160 = −3.091; p = 0.002, d = 0.473), MABC-2-C A–Static , MABC-2-C A1 , MABC-2-C A2 , MABC-2-C A3  and MABC-2-C B3 . MABC-2-C B–Dynamic, B1 and B2 did not show significant differences. All of these comparisons resulted in low (0.3) to medium (0.5) effect sizes. These significant differences were adjusted for sex and age by applying ANCOVA (p > 0.05). Considering the preterm ELBWs, preterm VLBWs and controls, we obtained significant differences for MABC-2-C A – Static  and MABC-2-C A1 . Bonferroni's post-hoc analysis revealed that MABC-2-C A–Static differences were only between the ELBW and control groups (p = 0.036), whereas MABC-2-C A1 differences were obtained between the ELBWs and controls (p = 0.030), and between the VLBWs and controls (p = 0.034). These differences remained as statistically significant after controlling for age and gender in the ANCOVA.Significant differences were found between preterm children and controls in the MABC-2-C total score (t3.3.p > 0.05), we considered the group of preterm children as a whole, without taking into account their birth weight, for the rest of the analyses. Thus, the correlation analysis yielded significant associations between the MABC-2-C total score and the corrected age of sitting , crawling  and walking . MABC-2-C A–Static was significantly associated with the age of sitting , crawling  and walking . MABC-2-C-B–Dynamic did not show any significant association with the milestones. Significantly correlated variables were included in a stepwise regression model, and only crawling was entered in the model, predicting the outcome of the MABC-2-C total score (R2 = 0.483).First, we calculated the Pearson correlations between the different measures of MABC-2-C and the birth weight of the preterm children. As we did not obtain any statistically significant correlation (3.4.p = 0.014), primary apnea , days of ventilation  and intraventricular hemorrhage grade . MABC-2-C A–Static correlated significantly with the use of prenatal steroids , patent ductus arteriosus , apnea , days of ventilation  and intraventricular hemorrhage grade . MABC-2-C B–Dynamic did not show any association with perinatal variables. Significantly correlated variables were included in a stepwise regression model. Intraventricular hemorrhage, the use of prenatal steroids and the days of ventilation were entered in the model, predicting the outcome of the MABC-2-C total score (R2 = 0.227).Correlation analyses were performed to identify significant perinatal variables with the scales of motor assessment. The results showed that the MABC-2-C total score was significantly correlated with the use of prenatal steroids  Considering birth weight, the ELBWs and VLBWs obtained lower scores than the controls on Scale A1, which is related to personal autonomy behaviors. Functional difficulties related to self-care have been previously reported in preterm children In the preterm group in general, the age at which they achieved sitting, crawling and walking was related to their motor development in later childhood, but only the age of crawling was a significant predictor of such subsequent motor development. Previous research has found associations between motor milestones, i.e., grasping an object The age of crawling being a significant predictor of later motor development has some limitations. First, a high percentage of the preterm and term-born children in the sample did not crawl. Second, there were fewer preterm children who crawled than controls. Therefore, it is difficult to consider crawling as a "universal" predictor of later development, given that the absence of crawling is common within development; yet, if a preterm infant starts to crawl later, this may be a warning sign for subsequent motor development in infancy.Later psychomotor development was associated with apnea, prenatal steroid use, days of mechanical ventilation and intraventricular hemorrhage, the latter three factors being significant predictors of later motor development. Coinciding with our findings, events involving early brain damage, such as intraventricular hemorrhage, pose a greater risk for poorer motor development in later childhood A limitation of our study is that our findings were obtained from the checklist and not from the complete MABC-2 evaluation. In this regard, there is a marked absence of studies that relate the checklist or questionnaire scores to performance-based tasks. Other questionnaires widely used for the diagnosis of DCD, such as the Developmental Disorder Coordination Questionnaire (DCDQ) 5.In conclusion, ELBW and VLBW preterm children may present motor developmental disturbances in childhood, mainly those related to personal autonomy. The age of attainment of certain motor milestones in premature children may be related to later motor performance, without necessarily implying a delay in the age of acquisition. Likewise, certain perinatal factors related to early cerebrovascular events and respiratory difficulties are also associated with motor performance, probably because they involve an alteration in later brain development. In this sense, screening tools could be used for the detection of a possible case of motor developmental risk in regular pediatric consultations.


The peanut group was advised to consume 35 g of lightly salted dry-roasted peanuts prior to two main meals per day. Participants in the control group were given education to follow a low-fat diet. Both groups had dietetic counseling to restrict energy intake . Outcome assessment occurred at baseline, 3 months, and 6 months. In total, 107 participants were randomized , and 76 participants completed the study. No between-group difference in body weight (primary outcome) was observed at 6 months . The mean weight loss at 6 months was 6.7 ± 5.1 kg in the cohort (visit p < 0.001). HbA1c, fasting glucose, fasting insulin, 2-h glucose, and HOMA-IR were not different between the groups. Systolic blood pressure was reduced to a greater extent in the peanut group vs. the control group at 6 months . Intake of 35 g of peanuts prior to two main meals per day, in the context of an energy-restricted diet, resulted in weight loss comparable to a traditional low-fat weight loss diet without preloads. Greater systolic blood pressure reductions were observed with peanut intake, which may lower cardiovascular disease risk.The objective of this study was to examine the effect of consuming 35 g of peanuts prior to two main meals per day as part of a weight loss diet, compared to a traditional low-fat weight loss diet, on body weight, markers of glycemic control, and blood pressure in adults at risk of type 2 diabetes over 6 months. A two-arm randomized controlled trial was conducted. Adults (age > 18 years) with a BMI of >26 kg/m Overweight and obesity continues to be an issue of global public health significance. In the United States, approximately 74% of adults aged >20 years have overweight or obesity . Similarp < 0.05) to a greater extent than an isocaloric lower-protein fiber-based shake  after 84 days [The first-line intervention for treatment of overweight and obesity is an energy-restricted diet ; however 84 days . In addi 84 days ,8,9. Oil 84 days . Importa 84 days . TherefoSubstantial evidence shows that nuts are associated with a lower risk of CVD and type 2 diabetes . These fA 6-month 2-arm parallel randomized controlled trial was conducted at the University of South Australia, Adelaide, Australia, to examine the effect of an energy-restricted diet including 70 g/d of peanuts on weight loss, blood pressure, and glycemic outcomes compared with a low-fat weight loss diet. The peanut group was advised to consume 35 g of lightly salted dry-roasted peanuts prior to two main meals per day. Participants in the control group were given education to follow a low-fat diet. Both diet groups were advised to restrict energy intake . Participants were randomized at baseline in a one-to-one ratio, using a computer-generated scheme (randomization.com). The study was approved by the University of South Australia Human Research Ethics Committee, and written informed consent was obtained from the participants . The study was conducted in accordance with the Declaration of Helsinki.2, and were at moderate or high risk of type 2 diabetes (score > 6 points), as assessed by the Australian type 2 diabetes risk assessment tool (AUSDRISK) [Participants were recruited from January 2021 to May 2021 from Adelaide, Australia, using print, social media, and radio advertising. Eligible individuals were >18 years of age, had a body mass index (BMI) >26 kg/mBoth the peanut and control group received nutrition education from an accredited practising dietitian to follow an energy-restricted diet. Participants in both groups met with the dietitian monthly throughout the study. Based on previous studies, women and men were counseled to restrict energy intake to 5500 and 7000 kJ, respectively ,22. PartParticipants in the peanut group were provided education to eat 35 g of peanuts 30 min prior to two of their meals  for the entire 6-month study period. Lightly salted dry-roasted peanuts  were provided for the duration of the study. Intake of the provided peanuts was assessed by a daily checklist completed by the participants. Participants in the control group were given education to follow a low-fat diet and asked to avoid peanuts and peanut butter for the duration of the study. Dietetic education to follow an energy-restricted diet is reflective of standard care for management of overweight and obesity . ParticiParticipants attended the research center on 7 occasions . At baseA single non-random 24 h recall was collected at baseline, 3 months, and 6 months, using the Automated Self-Administered 24 h Recall (ASA-24) System . Completion of a single 24 h recall at each timepoint is recommended for assessing changes in mean usual intake in response to an intervention . Particip < 0.05) [Sample size calculations showed that the completion of 50 participants in each group would provide 80% power to detect a 1.7 kg (standard deviation 3.0 kg) difference between the groups ( < 0.05) . Weight All the statistical analyses were performed with SAS . All available data from randomized participants were included in data analyses consistent with intent-to-treat principles. Data from participants who withdrew from the study were included when endpoint measures were obtained. The mixed-models procedure does not perform listwise deletion, thus preserving the degrees of freedom; therefore, this analytical approach allowed for inclusion of participants with ≥1 missing data point. The normality of the residuals was assessed by using univariate analysis (PROC UNIVARIATE) to quantitatively evaluate skewness and to visually inspect the distribution and normal probability (Q–Q) plots.p-value. Sex effects and sex-by-diet interactions were also evaluated. Statistical significance was set at p < 0.05.The mixed-models procedure (PROC MIXED) was used to examine the effect of diet on each outcome. Visit was modeled as a repeated effect to account for the repeated-measures design. Diet was modeled as a fixed effect, and the baseline value was included as a covariate. When a main effect of diet, visit, or diet by visit was detected, post hoc pairwise comparisons were conducted and the Tukey–Kramer method was used to adjust for multiple comparisons; data from post hoc testing are presented as the pairwise mean difference and 95% CI with the Tukey–Kramer adjusted 2, and waist circumference of 109 ± 12.9 cm  for weight; no diet effect (p = 0.94) or visit-by-diet interaction (p = 0.98) was observed (p = 0.92). No sex effects or sex-by-diet interactions were observed. Only three participants in each group did not lose weight at 6 months compared to the baseline.A visit main effect was observed (observed . Comparep = 0.007) and visit (p < 0.001) were observed; the diet-by-visit interaction (p = 0.063) approached statistical significance (p < 0.001) and the control group  after 6 months. The 6-month reduction in systolic blood pressure observed in the peanut group was significantly greater than the corresponding change observed in the control group . No sex effects or sex-by-diet interactions were observed for systolic blood pressure.For systolic blood pressure, main effects of diet (ificance . Comparep < 0.001) and 6  months compared to baseline. No difference in diastolic blood pressure was observed between 3 and 6 months. No sex effects or sex-by-diet interactions were observed.No diet effect or diet-by-visit interaction was observed for diastolic blood pressure. A visit main effect was observed for diastolic blood pressure. Diastolic blood pressure declined in the cohort at 3 . Compared to baseline, fasting glucose was lower at 3 months  and 6 months  in the cohort. No sex effect or diet-by-sex interaction was observed for fasting glucose. No main effects of visit, sex, or sex-by-diet were observed for 2-h glucose.No diet effects or diet-by-visit interactions were observed for fasting glucose, fasting insulin, 2-h glucose, HbA1c, or HOMA-IR . Fastingp < 0.001). Compared to baseline, insulin was lower at 3 months  and 6 months . No sex effect or diet-by-sex interaction was observed for insulin. HOMA-IR also declined over time in the whole cohort (p < 0.001). Compared to baseline, the HOMA-IR was lower at 3 months  and 6 months . No sex effect or diet-by-sex interaction was observed for HOMA-IR.Insulin declined over time in the whole cohort (visit p < 0.001). Compared to baseline, HbA1c was lower at 3 months  and 6 months  in the whole cohort. HbA1c was also lower at 6 months compared with 3 months . A sex effect (p = 0.03) was observed, whereby women had higher HbA1c vs. men; however, no diet-by-sex interaction was observed.HbA1c declined in the cohort over time (visit p = 0.01) compared to the peanut group; no between-group difference was observed at 3 months. The percentage of energy from total fat was significantly higher in the peanut group vs. the control group at 3 months  and 6 months . The higher intake of fat in the peanut group was explained by the higher intake of MUFA from the provided peanuts. Compared to the control group, energy intake from MUFA was higher in the peanut group at 3 months  and 6 months . The percentage of energy from carbohydrates was significantly lower in the peanut group vs. the control group at 3 months  and 6 months . These data confirm a high compliance level in both groups since the differences reflect intake of a high fat food  vs. the low-fat diet (higher in carbohydrates).Main effects of diet, visit, and diet-by-visit were observed for intake of energy, total fat (g and % kJ), MUFA (% kJ), and carbohydrates (% kJ) . Post hop = 0.004). A visit effect was also observed for sodium. In the whole cohort, sodium intake was lower at 3 months  and 6 months  compared to the baseline; no diet effect or diet-by-visit interaction was observed for sodium intake. Main effects of diet and diet-by-visit were observed for potassium. Potassium intake was higher in the peanut group vs. the control group throughout the study. Post hoc testing showed no significant difference in potassium intake between the groups at each timepoint . A diet-by-visit interaction was observed for fiber; post hoc testing showed no significant difference in fiber intake between the groups at each timepoint .A visit effect was observed for saturated fat, and post hoc testing showed that intake was lower at 3 months compared to baseline in the whole cohort  and concern has been raised about the potential for habitual intake of nuts to promote weight gain . For thiIn the present study, we observed greater systolic blood pressure lowering (−5 mmHg) in the peanut group compared to the control group at 6 months. Based on a recent meta-analysis, a 5 mmHg reduction in systolic blood pressure would be expected to lower the risk of a major cardiovascular event by 10% . In indiA meta-analysis of 21 randomized controlled trials showed that nut intake lowered systolic blood pressure in individuals without type 2 diabetes ; however, only two of the included studies examined peanuts, and no effect on systolic blood pressure was observed in both studies . A more The higher-MUFA/lower-carbohydrate intake in the peanut group may have contributed to the observed systolic blood pressure lowering. A systematic review and meta-analysis of randomized controlled trials including patients with type 2 diabetes showed that the intake of a high MUFA diet lowered systolic blood pressure  compared to a high carbohydrate diet . HoweverIn both groups, sodium intake was reduced over the 6-month period, which likely contributed to the systolic blood pressure reductions observed over time. However, the reduction in sodium intake would be expected to lower systolic blood pressure by <1 mmHg based on a meta-regression showing systolic blood pressure lowering of 0.042 mmHg per 1 mmol reduction in sodium excretion per day . PotassiIn this study, we did not observe any between-group differences in fasting glucose or insulin, HOMA-IR, 2-h glucose, or HbA1c. A meta-analysis of randomized controlled feeding studies showed that replacement of 5% of energy from carbohydrates with MUFA had no effect on fasting glucose, 2-h glucose, or fasting insulin . HoweverIt is also plausible that the hypothesized improvements in glycemic control were not observed because the MUFA that was consumed as part of the peanut matrix had limited intestinal bioavailability, and therefore did not delay gastric emptying, reduce carbohydrate absorption, and/or stimulate insulin secretion to lower postprandial glucose excursions, which has been observed with the intake of MUFA-rich oil prior to a meal . Given tThis study has several strengths, including the randomized controlled design, the 6-month follow-up period, and the provision of nutritional counseling by a dietitian. However, this study is limited by the lack of assessment of 2-h insulin concentration, as well as measurement of insulin sensitivity. Characterization of changes in insulin sensitivity would provide insights into the effect of the diets on reversing insulin resistance and delaying type 2 diabetes. In addition, we did not assess waist circumference or loss of lean and fat free mass following the weight loss diets. The control group in this study was provided with dietetic education to follow an energy-restricted diet, which is reflective of standard care for the management of overweight and obesity. However, since the control group did not consume a preload, no inferences can be made about the superiority of a peanut preload vs. other preloads. Finally, attrition in the control group was greater than in the peanut group, which may have affected our power to detect statistically significant differences in the primary outcome between the groups. However, based on the effect observed and the 95% CI  it is unlikely that there was a clinically significant difference between the groups.In conclusion, intake of 35 g of lightly salted dry-roasted peanuts prior to two main meals per day, in the context of a weight loss diet, resulted in similar weight loss to a traditional low-fat weight loss diet in adults at high risk for type 2 diabetes after 6 months. No differences in HbA1c, fasting glucose, fasting insulin, or 2-h glucose were observed between the two weight loss diets. Greater reductions in systolic blood pressure were observed with the peanut-containing weight loss diet, which may lower CVD risk.


MEFV, which lead to a pro-inflammatory state and increased production of Interleukin 1 beta subunit (IL-1b) by myeloid cells. Despite the overall positive results obtained with anti-IL-1 agents in FMF patients, little is known about the long-term growth impact of these drugs in the pediatric population.Familial Mediterranean Fever (FMF) is the most common monogentic autoinflammatory disease. FMF results from mutations in To assess the long-term body weight and height trajectories in children with FMF treated with anti-IL-1 agents.We conducted a retrospective analysis of 646 pediatric FMF patients followed in our center, of whom 22 were treated with either anakinra (36.3%) and/or canakinumab (90.9%). Patients were assessed for demographic, clinical and genetic characteristics and were followed for a mean of 3.05 ± 1.75 years. Data of height and weight percentiles were recorded before and after treatment.p < 0.001). Overall, anakinra and canakinumab resulted in a complete response in 80% of patients and exhibited low rates of adverse effects. We found a significant increase in height and body weight percentiles following treatment .The most common indication for IL-1 blockers treatment was colchicine resistance (66.6%). Ninety percent of those patients had a moderate or severe disease according to the Pras score and had higher proportion of M694V homozygosity compared with patients who did not require anti IL-1 agents (95.2% vs. 30.5%, Treatment with anti-IL-1 agents in children with FMF is effective and safe and may potentiate long-term growth. MEFV gene located on chromosome 16 which encodes pyrin, a 781-amino acid long protein expressed primarily in cells of the innate immune system. There, it serves as a negative regulator of a cytosolic cellular multiprotein complex termed the inflammasome, which mediates the activation of various inflammatory pathways in response to deleterious cellular insults  and drives the maturation of the IL-1b and IL-18. Accordingly, dysregulated inflammasome activity results in a pro-inflammatory state and has been linked to autoinflammatory diseases [MEFV, the most common is M694V, which has been shown to present in 20–65% of patients. Importantly, M694V homozygotes suffer from a more severe form of the disease, including frequent episodes and the need for higher colchicine doses to prevent attacks, as compared to patients with other mutations [MEFV gene [Familial Mediterranean Fever (FMF) represents the most common form of monogenic autoinflammatory disease . It is cdiseases . In FMF,diseases , 3. AmonEFV gene –12. ThesEFV gene . In receEFV gene , and canEFV gene . Both drEFV gene . DespiteMEFV, for those who had undergone genetic testing. Patients were followed until the end of the study (2022) or until treatment cessation . We also extracted any documented side effects, based on description of symptoms, physical examination, and/or laboratory tests.We carried out a historical cohort study by collecting and analyzing data of 646 pediatric FMF patients treated at Sheba Medical Center in Israel between the years 2006–2022, including 22 patients who were treated with anti-IL-1 agents, anakinra and/or canakinumab Patients were started Anakinra only when Canakinumab was not approved by their health insurance as their first biologic agent. All patients were diagnosed according to Tel-Hashomer criteria . DemograWe classified the response to treatment as complete response , partial response  and no response.Disease severity was calculated according to Pras severity criteria . Pras Scp-value < .05 was considered significant. All statistical analyses were calculated using prism graphpad v.9.Results are presented as mean ± standard deviation or as proportion (%) as indicated. Differences between the groups in discrete variables were evaluated by Chi-square test. Comparison of genetic variants between groups was conducted using the Fisher Exact test. Paired Student t test was used to compare pre and post treatment differences in continuous variables (height and weight percentiles). Normality of body weight and height was verified using D’Agostino and Pearson test. A two-tailed This study was approved by the Institutional Review Board of Sheba Medical Centre.We retrospectively collected data from a cohort of 646 pediatric FMF patients treated at our Center between the years 2006–2022. Among these, 22 patients (3.4%) required treatment with an anti-IL1 agent. Over the years of follow-up, 20 (90.9%) of whom were treated with canakinumab and 8 (36%) were treated with anakinra (Table MEFV genotype. This included 173 (33.4%) patients who were homozygous, 145 (28%) who were compound heterozygous, 172 (33.2%) who were heterozygous and 27 (5.2%) individuals in whom no mutation was identified. Mutation analysis was available for 21 out of the 22 patients who were treated with anti-IL-1 agents. Thus, we compared the mutation’s distribution between these 21 patients to our genetically identified FMF cohort. As indicated in Table p < 0.0001). In fact, 12.6% of the patients harboring M694V homozygous mutations were eventually treated with anti-IL-1 agents. One additional patient in the anti-IL-1 group was a compound heterozygote for M694V/V726A.Of the 646 pediatric FMF patients in our cohort, 517 patients had available information regarding their ous, 145 8% who we and height  percentiles following treatment initiation , with 56 and 75% of treated children demonstrating an increase in body weight and height percentiles at the end of the follow-up, respectively. We next conducted an additional analysis comparing growth percentiles in the year before initiation of biologic treatment, while all patients were actively treated with colchicine, with baseline growth parameters. As demonstrated in Fig. Overall, 80% of patients treated with these agents demonstrated complete response, with an additional 20% showing partial response with none had no response Table , two patFig. A and height Fig. B percentLastly, we assessed the safety of anti-IL1 treatments in our cohort. Overall, during 771 patient months of follow-up, one patient reported a local skin reaction at the injection site, and two patients complained of abdominal pain. Notably, no additional side effects were noted.Anti-IL-1 agents are highly effective and safe in patients with colchicine-resistant FMF , 21, 22.The main finding of our study is the significant increase in both height and body weight percentiles among FMF patients treated with anti-IL-1 drugs. Our results corroborate with historical reports demonstrating a positive effect of colchicine on growth and development in pediatric FMF patients –12. Of iTwo previous studies reported the influence of canakinumab on linear growth in autoinflammatory conditions in children. In the study of Balci et al. of 11 children with FMF, despite a median follow-up of only 1.5 years, canakinumab treatment had a positive effect on both height and body weight Z-scores . Yücel eMEFV mutations in the various cohorts.There are different reports in the literature regarding the percentage of patients who had complete response to anti IL-1 agents. Complete remission following canakinumab treatment was reported by several groups, such as Brik et al. who reported 42.9% with complete response , De BeneInterestingly, in this study we report a very low rate of side effects, despite a mean follow-up of more than 3 years. One patient reported an injection site skin reaction, and two additional patients complained of abdominal pain. These results are in-line with published data regarding canakinumab safety , 22, 27.As for the genetic profile of pediatric FMF children, our findings are consistent with the more severe phenotype described in patients who are homozygous for the M694V mutation . We founThis study has several limitations that require consideration. First, the retrospective design does not allow us to infer on causality. Second, the relatively small number of patients limits our ability to address the age and sex specific effects of anti-IL-1 drugs on growth. Yet, our comprehensive database with detailed clinical parameters of over 600 FMF patients treated at our institution and a mean follow-up of over 3 years provide us with a unique opportunity to conclude regarding the long-term anthropometric effects of anti-IL-1 agents in children with FMF.Our study shows that anti -IL-1 agents represent an effective and safe treatment and may improve growth in children with colchicine-resistant FMF. Therefore, we suggest considering early treatment when needed, especially for those who harbor the M694V homozygous mutation.


Univariate and multivariate logistic regression analyses were performed to assess the early mortality rate. The final model consisted of hsa-miR-409-3p, hsa-miR-328-3p, age, and R-ISS 3. It yielded an area under the curve (AUC) of 0.863 (95%CI: 0.761–0.965) with 88.2% sensitivity and 77.5% specificity. Further external validation of our model is needed to confirm its clinical value.Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM) microenvironment. Despite the progress made in treatment, some MM patients still die within the first year of diagnosis. Numerous studies investigating microRNA (miRNA) expression patterns suggest they may be good prognostic markers. The primary aim of this study was to analyze the expression of selected miRNAs in the serum of MM patients who were later treated with bortezomib-based regimens, and to determine their potential to predict early mortality. The study was conducted in 70 prospectively recruited patients with newly diagnosed MM admitted to the Department of Hematology of the Copernicus Memorial Hospital, Lodz (Poland) between 2017 and 2021. Among them, 17 patients experienced death within 12 months of diagnosis. The expression of 31 selected miRNAs was determined using a miRCURY LNA miRNA Custom PCR Panel. The obtained clinical data included patient characteristics on diagnosis, treatment regimen, response to treatment, and follow-up. Differential expression analysis found two miRNAs to be significantly downregulated in the early mortality group: hsa-miR-328-3p (fold change—FC: 0.72,  Multiple Myeloma (MM) is a malignant, heterogenous disease characterized by the clonal expansion of antibody-producing plasma cells, mostly with their origin in the bone marrow. In addition, symptomatic MM is associated with hypercalcemia, renal impairment, anemia, lytic destruction of bones (the ‘CRAB’ criteria) . MultiplDespite significant improvements in MM treatment modalities, a subset of patients still experiences abbreviated responses and short survival ,7. TreatAn increasing number of studies underline the role of miRNAs in the pathogenesis of MM and their potential role in its diagnosis and prognosis ,28. It wThe study was performed in 70 previously untreated MM patients with a mean age at diagnosis of 65 ± 10.97 years including 34 men and 36 women. Among them, 17 patients experienced death within 12 months of diagnosis. All early deaths were MM-related . Demographic, clinical and laboratory characteristics of the 70 MM patients enrolled for the study can be found in p = 0.0007) and male sex (p = 0.04). No differences in R-ISS distribution or CRAB symptoms were found between the groups. Similarly, no significant trends in percentage of myeloma infiltration in the bone marrow were noted regarding laboratory results, including LDH, serum M protein, albumin, CRP, and uric acid. Early mortality was significantly associated with older age  and hsa-miR-409-3p . A volcano plot of miRNAs with significant differences in expression is shown in In differential expression analysis, two miRNAs were significantly downregulated in the early mortality group—hsa-miR-328-3p  and hsa-miR-409-3p  were acting protectively in relation to the occurrence of early mortality. The results of univariate logistic regression analyses for all miRNAs can be found in Univariate logistic regression analyses were performed to assess the early mortality occurrence. Similarly, hsa-miR-328-3p , hsa-miR-328-3p  and age . The combination of hsa-miR-409-3p, hsa-miR-328-3p, and age at diagnosis could discriminate patients with high risk of early mortality.A multivariate logistic regression analysis was also performed. A forward stepwise and backward stepwise selection approach were used to restrict the model to the most predictive miRNAs together with clinical features. Forward stepwise selection yielded a final model with the lowest AIC value. In the multivariate logistic regression analysis, the two miRNAs retained their significance. The final model  consisteA receiver operating characteristics (ROC) analysis for the model yielded an area under the curve (AUC) of 0.84 (95%CI: 0.73–0.95). The ROC curve is shown in p = 0.001) with its lower expression in the R-ISS I group compared to R-ISS II (p = 0.015) and R-ISS III group (p = 0.001). A box plot of the hsa-miR-627-5p expression in different R-ISS groups can be found in p = 0.004, In the analysis of different cytogenetics risk groups, it was found that the expression of hsa-miR-627-5p was significantly different between R-ISS groups , which combines albumin and β2-microglobulin levels, and the presence of chromosomal abnormalities detected via FISH (fluorescence in situ hybridization) ,33. HoweManier et al. found that let-7b and miR-18a were significantly associated with both progression-free survival (PFS) and overall survival (OS) in univariate analysis and remained statistically significant after adjusting for the ISS and adverse cytogenetics in multivariate analysis . Hao et Following on from our previous study examining the value of serum microRNA expression signature in predicting refractoriness to bortezomib-based therapy in multiple myeloma patients , the preIt was reported that miRNA-328 may decrease chemoresistance in glioblastoma cancer cells and breast cancer cells by down-regulating the ABCG2 gene ,50. DownAlthough the risk of MM increases with age, no data could be found about the association between hsa-miR-409-3p or hsa-miR-328-3p expression and age or senescence in existing literature. The role of these miRNAs in the pathogenesis of multiple myeloma to date also remains unknown. However, hsa-miR-627-5p has been investigated in other cancers including hepatocellular carcinoma and colorectal neoplasms ,55. Our The study was performed in previously untreated MM patients who were qualified for treatment with bortezomib-based regimens based on the decision of the medical board. Sets of clinical and laboratory data were obtained: patient characteristics on diagnosis, treatment regimen, response to treatment and follow-up. For inclusion in the study, the patients needed to have newly diagnosed, untreated multiple myeloma, and measurable disease confirmed by the presence of monoclonal protein in blood or Bence–Jones proteinuria. Patients were treated at the Department of Hematology, Copernicus Memorial Hospital, Lodz, Poland in the years 2017–2021. Responses to treatment, progression free survival (PFS), and overall survival (OS), were assessed according the International Myeloma Working Group (IMWG) criteria ,57. Earlg for 10 min, as described previously [Venous blood samples were collected from previously untreated MM patients in serum separating tubes. The samples were processed within two hours of collection by centrifugation at 2400× eviously . Serum sThe cDNA was synthesized from the obtained total RNA including mature miRNAs (<200 bp), using the miRCURY LNA Reverse Transcription Kit , according to the guidelines provided by the manufacturer. Mature miRNAs were polyadenylated by poly(A) polymerase and reverse transcribed into cDNA using oligo-dT primers. Polyadenylation and reverse transcription were performed in parallel in the same tube. The oligo-dT primers have a 30-degenerate anchor that allows the amplification of mature miRNA in the real-time PCR step. The total volume of 2 µL of the isolate was added to the reaction tube containing 2 µL of 5× miRCURY RT Reaction Buffer, 1 µL of 10× miRCURY RT Enzyme Mix and filled with RNase-free water to make up a final volume of 10 µL reaction mix. Two synthetic miRNAs: UniSp6 (0.075 fmol) and cel-miR-39-3p (0.001 fmol) were used as a positive control for cDNA synthesis. The reaction took place at 42 °C for 60 min, followed by inactivation at 95 °C for 5 min using a TProfessional thermal cycler . The cDNA was stored at −20 °C until further use.Before the miRNA expression analysis, the RNA samples were subjected to quality control using the miRCURY LNA miRNA QC PCR Panel , according to the manufacturer’s protocol. The Panel contains 12 predefined assays for miRNAs that are expressed in a wide range of sample types used for evaluating samples in a biological context. Two of the miRNAs in the panel (miR-451 and miR-23a) were used to evaluate the level of hemolysis in serum samples. If the ΔCq (miR-23a—miR-451) is close to or higher than 7, there was an increased risk that the samples are affected by hemolysis, and these samples were rejected from the further analysis  . Only thp value significance from the univariate logistic regression analysis for overall survival . The selection of miRNAs included in this study was performed based on our previous analysis of 752 miRNAs  and was lts from ). The mi® Green I: 494 nm and 521 nm, respectively) was measured after each cycle. Melting curve analysis was performed after each qPCR to assess the specificity of the amplification. Absolute quantification of miRNA was determined using the LightCycler® 480 Software, Version 1.5 . Relative quantification of miRNA was determined by comparative Ct method.A premix of 3 µL of cDNA template (diluted 1:30), 5 µL of 2X miRCURY LNA SYBR Green PCR Master Mix and filled with RNase-free water to the final volume of 10 µL, was aliquoted into the PCR plate. Real-time PCR was performed on a LightCycler480 II Real-Time PCR System . The reaction was performed at 95 °C for 2 min, followed by 55 amplification cycles at 95 °C for 10 s and 56 °C for 1 min. Fluorescence signal . These miRNAs, according to the NormiRazor tool, proved to be the second most stable normalization pair in the currently analyzed group of patients and was chosen on the basis of our previous study where a triplet of miR-23b-3p, miR-151a-5p and miR-152-3p was chosen for the whole panel . Howevert-test when the data was normally distributed, and with the Wilcoxon–Mann–Whitney test when it was not, or if the variable was ordinal. These statistical tests were also used for the differential expression analysis comparing the normalized expression values in two experimental groups.The study group and intragroup association were described based on assumption testing using the Chi-squared or Fisher’s exact test. The normality of the distribution of continuous variables was verified with the Shapiro–Wilk test. For continuous variables, the difference between two groups was evaluated using a two-sided independent Student’s t-test was used to preselect variables for the development of the classification model. The miRNAs significant in the DE analysis were chosen as candidate variables in this step. Both a univariate model for each of the selected miRNA and a multivariate model that included all selected predictors were estimated. Forward stepwise and backward stepwise selection approaches were used to restrict the model. The lowest AIC (The Akaike information criterion) value was chosen as the determinant of the best final model. Receiver operating characteristics (ROC) and area under the curve (AUC) analysis were performed to determine the predictive power of the model and its ability to accurately predict early mortality.For a more comprehensive analysis, a logistic regression model was generated. The early mortality was used as outcome and the clinical features  together with miRNAs expression as predictors. The Student’s t-tests were used to compare the miRNA expression between different R-ISS groups depending on normality and homogeneity of variance. The Wilcoxon–Mann–Whitney test and the Student’s t-test were used to compare the miRNA expression between high-risk cytogenetics and standard-risk patients [The Kruskal–Wallis test with Bonferroni post hoc Wilcoxon–Mann–Whitney tests and the one-way ANOVA test with Bonferroni post hoc patients ,6,7. p values lower than 0.05 were considered statistically significant. As all the analyses were preplanned, no correction for multiple comparisons was applied.All statistical analyses were conducted using Statistica Version 13.1  and R programming language (version 4.1.3) software. The OmicSelector R package was partly used in the analysis  p valuesIn conclusion, we identified two miRNAs, hsa-miR-409-3p and hsa-miR-328-3p, that are independent factors related to early mortality in MM patients. We used our results to generate a multiple regression model that may have the potential to predict early mortality. However, further study is needed to confirm our findings.